Longitudinal growth of limbs occurs through cartilaginous structures called the growth plate at the ends of each bone in a process called endochondral bone formation. One important feature of the growth plate is that the cells in the tissue align into columns. While the magnitude of bone growth is dependent on cell proliferation, matrix deposition, and cell enlargement during hypertrophy, the columns allow directional growth of the bone. It was noted that a large proportion of children with paralysis in one leg as a result of poliomyelitis or hemiplegic cerebral palsy demonstrated significant limb length discrepancy with the paralyzed limb being shorter than the other limb. Based on these observations we hypothesized that mechanical load regulates the function of the growth plate. While the effects of mechanical load on bone remodeling have been studied extensively, little was known about the role of loading on endochondral bone formation and limb length determination so we developed in vivo models for removing mechanical load on hind limb in young mice via paralysis. Loss of mechanical load resulted in shortening of the paralyzed limb, disorganization of the columnar architecture in the growth plate, and disruption to the cortical actin structure within the cells. Very little is known about how chondrocytes align themselves into this columnar structure because isolated chondrocytes in culture do not align into columns and in vivo models are time consuming and expensive to work with. In addition, there are limited methods to view the biological processes involved in real time. In this R21proposal we plan to address a critical barrier in the field and develop an ex vivo organ culture system and live cell imaging assays to measure changes in protein localization and tension at cellCcell and cellCmatrix adhesion sites during column formation in real time. We will then illustrate how the mechanisms involved in column formation in loaded and unloaded conditions can be analyzed in detail using these assays. Understanding the molecular mechanisms that govern how mechanical load affects growth plate function would be expected to inform future strategies for treating various types of limb length disorders.